Lipoproteins are the primary carriers of plasma cholesterol and triglycerides. They are micellar lipid-protein complexes that contain protein (referred to as apoprotein) and polar lipids organized in a surface film that surrounds a neutral lipid (triglyceride and cholesteryl ester) core. Lipoproteins were originally identified based on their bouyant densityies as measured by ultracentrifugation. Accordingly there are four major density classes: chylomicrons, very low density lipoproteins (VLDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL).
Paralleling advances in the technology of ultracentrifugal separations there has been a further subdivision of the LDL and HDL density classes into further subclasses of greater homogeneity. For instance, LDL can be resolved into an intermediate density lipoprotein (IDL) and an LDL.sub.2 subclass. However, even these subclasses are composed of functionally heterogeneous populations of lipoprotein particles because of their varied apoprotein content.
Eight major apoproteins, A-I, A-II, A-IV, B, C-I, C-II, C-III, and E, have been isolated, and of the group of minor apoproteins that can be recovered in larger amounts from certain density classes, most can also be found in other density classes. Thus, most LDL particles contain only apo B, however a few particles also contain other apoproteins and this accounts for the trace amounts of apo C-I, apo C-II, C-III, and apo E present in this density class.
In some cases, specific functions have been assigned to particular apoproteins. For instance, a species of apo B synthesized in the liver, termed apo B-100, is recognized and bound by cellular LDL receptors. By binding apo B-100, these receptors bind LDL particles and extract them from the plasma. The LDL is thereby taken into the cells and broken down, yielding its cholesterol to serve each cell's needs. The apo B-LDL receptor interaction thus plays a major role in removal of LDL cholesterol from the bloodstream.
Another species of apo B, termed apo B-48, is not recognized by the LDL receptor. This apo B species, which is only 48 percent as large as apo B-100, is synthesized in humans only by the intestine. Lipoproteins containing apo B-48, such as chylomicrons and chylomicron remnants, do not bind to the LDL receptor.
Although these two species of apo B appear to be under separate genetic controls (a single patient has been described whose body makes apo B-48, but not apo B-100), immunologic studies have demonstrated that apoproteins B-100 and B-48 share antigenic determinants. At least three research groups have reported generation of a total of seven different monoclonal antibodies that bind to either of apo B-100 and apo B-48. The data reported by those researchers strongly suggest that apo B-48 and apo B-100 are structurally related proteins; i.e., that apo B-48 may represent a portion of the apo B-100 protein. Evidence also has been reported that apo B-48 and apo B-100 are not found on the same lipoprotein particle, suggesting that separate apo B particles exist.
Recently, several investigators have suggested that plasma levels of apo B may be more predictive of coronary artery disease (CAD) risk than plasma LDL cholesterol levels. Sniderman et al., Proc. Natl. Acad. Sci. USA 77, 604-608 (1980). Because artherosclerotic vascular disease and its complications continue to be the leading cause of death and debilitation in Western society, there has been a long felt need within the biomedical industry for assay systems capable of identifying individuals at risk for CAD.
Many types of immunoassays for plasma apoprotein B utilizing specific antibody-containing antisera have been reported, including competitive fluid phase and solid phase radioimmunoassays (RIA), enzyme-linked immunosorbant assays (ELISA), radial immunodiffusion assays and others. Problems limiting the widespread application of these apo B immunoassays have been reproducibility, and the quality and specificity of the antisera used. Reviews of the methodological problems of each of the various types of apo B assays are found in Currey et al., Clin. Chem. 24, 280-286 (1978) and Rosseneu et al., Clin. Chem. 28, 427-433 (1983).
Several investigators have reported development of panels of monoclonal antibodies against human apo B for use in studying its antigenic structure and role in lipoprotein metabolism. Furthermore, there have been reports of using anti-apo B monoclonal antibodies to measure plasma apo B levels in fluid-phase RIA's. Patton et al., Clin. Chem. 29, 1898-1903 (1983) and Maynard et al., Clin. Chem. 30, 1620-1624 (1984). In addition, one group has reported use of a mixture of anti-apo B monoclonal antibodies in a radial immunodiffusion assay for plasma apo B. Marconvina et al., Clin. Chim. Acta 147, 117-125 (1985). However, these assay techniques suffer from the necessity of lengthy incubations, repeated centrifugation or use of radioactive materials.
The use of monoclonal antibodies as reagents for assaying for the presence of apo B-100 in human body fluid samples is attractive because once obtained, such reagents can be produced in relatively large amounts with consistent quality. However, there are a number of factors that militate against the use of a particular monoclonal antibody as a component in an apo B-100 assay system.
First, the art teaches that a monoclonal antibody can be too immunospecific to be useful because of the antigenic heterogeneity of its target antigen. For example, the specificity of conventional polyclonal antibody-containing antisera depends on a consensus of hundreds of thousands of different antibodies that bind to antigenic determinants covering most or all of an antigenic protein. As a result, small changes in the structure of the antigen due to genetic polymorphism, heterogeneity of glycosylation or slight denaturation will usually have little effect on polyclonal antibody binding. Similarly, a larger or smaller subset of antibodies from polyclonal antisera will usually bind antigens that have been modified or denatured.
In contrast, monoclonal antibodies usually bind to one antigenic determinant (epitope) on the antigen molecule. If, for any reason, that determinant is altered, the antibody may or may not continue to bind. Whether this is a problem or an advantage depends on the individual circumstances. If, as in the present case, the monoclonal antibody is to be used in a diagnostic assay for an apoprotein, a minor antigenic variation in that protein could cause gross errors.
The antigenic heterogeneity of apoprotein B-100 is well documented. For instance, epitope expression on apo B has been found to be modulated by (1) the composition of the associated lipids, (2) temperature of the immunoreaction, (3) the degree of isolation of LDL from its native environment, and (4) genetic expression between individuals.
Second, because of their unique specificity, the successful use of a monoclonal antibody (MoAb) is often dependent on its affinity for the target antigen. For instance, whereas a MoAb may have sufficient affinity to be useful in binding liquid and solid phase antigen while the MoAb is itself in liquid phase, that same antibody may not be useful as a solid phase-affixed antibody that is useful in binding to and "pulling" the antigen out of solution.
The above problems are generic to the use of monoclonal antibodies. Those skilled in the art have therefore recognized that it is essential to test and characterize monoclonal antibodies in any assay system in which they are to be used. See Goding, James W., Monoclonal Antibodies: "Principles and Practice." Pages 40-46, Academic Press, New York (1983).